Complement inhibition for improved nerve regeneration

ABSTRACT

The present invention relates to methods and medicaments used for treating conditions that require axonal regeneration, e.g. in mammals affected by injury or disease of the central or peripheral nervous system. The medicaments used in these methods facilitate axonal regeneration by inhibition of the complement system. Conditions requiring axonal regeneration that may be treated in accordance with the invention include physical injuries as well as neurodegenerative disorders of the peripheral or central nervous system.

FIELD OF THE INVENTION

The present invention relates to methods and medicaments used fortreating conditions that require axonal regeneration, e.g. in mammalsaffected by injury or disease of the central or peripheral nervoussystem. The medicaments used in these methods promote axonalregeneration by inhibition of the complement system.

BACKGROUND OF THE INVENTION

Axon degeneration occurs frequently in many types of chronicneurodegenerative diseases and in injuries to axons caused by toxic,ischemic, or traumatic insults. It may lead to separation of the neuronfrom its targets, resulting in loss of neuronal function. One model ofaxon degeneration is the self-destructive process observed at the distalportion of a transected axon upon injury, termed Wallerian degeneration(WD) as first described by Waller (1850). In the process of WD, if anerve fiber is cut or crushed, the part distal to the injury (i.e. thepart of the axon separated from the neuron's cell nucleus) willdegenerate. Because most neuronal proteins are synthesised in the somaand carried to the axon by specialised axonal transport systems,degeneration of the transected axons has long been thought to resultfrom starvation of necessary proteins and other materials. However, thediscovery of a spontaneously occurring mutant mouse strain,C57BL/Wld^(s), whose axons survived for as long as weeks aftertransection suggested that Wallerian degeneration involves an active andregulated auto-destruction program.

Indeed one of the most striking cellular responses during WD in theperipheral nervous system (PNS) is the proliferation and infiltration ofmacrophages (Bruck, 1997). Macrophages participate in a wide array ofcellular responses during WD. Once activated, they release factors thatare mitogenic for Schwann cells (Baichwal et al., 1988). The completionof WD relies on the phagocytic ability of macrophages to degrade myelinand axonal debris (Griffin et al., 1992). In addition, macrophages candegrade molecules inhibitory to axonal regeneration (Bedi et al., 1992)as well as release factors, such as interleukin-1 (IL-1), which canpromote axonal growth via the induction of neurotrophic factors such asnerve growth factor (NGF) (Lindholm et al., 1987).

The precise mechanisms responsible for macrophage recruitment during WDare not completely understood. One group of factors that may play a rolein macrophage recruitment and activation is the serum complementproteins. The importance of complement proteins immune-mediatedperipheral nerve injury has been investigated previously.

Mead et al. (2002) showed that C6 deficient PVG/c rats, unable to formthe membrane attack complex (MAC), exhibit neither demyelination noraxonal damage and significantly reduced clinical score in theantibody-mediated experimental autoimmune encephalomyelitis (EAE) modelfor multiple sclerosis when compared with matched C6 sufficient rats.However, levels of mononuclear cell infiltration were equivalent tothose seen in C6 sufficient rats. Mead et al. (2002) concluded thatdemyelination and axonal damage occur in the presence of Ab and requireactivation of the entire complement cascade, including MAC deposition.

Jung et al. (1995) disclosed that treatment with recombinant humansoluble complement receptor type 1 (sCR1) markedly suppressed clinicalsigns of myelin-induced experimental autoimmune neuritis (EAN) in Lewisrats (an animal model of the human Guillain-Barré syndrome). Extendeddemyelination and axonal degeneration were also prevented. Thesefindings underscore the functional importance of complement duringinflammatory demyelination in the peripheral nervous system.

Indeed, in EAN, complement depletion diminished myelin breakdown andmacrophage recruitment in vivo (Feasby et al., 1987; Vriesendorp et al.,1995). Other groups have suggested that inhibition of the complementcascade reduces damage in neurodegenerative disease of the centralnervous system (CNS) (e.g. Woodruff et al. 2006; Leinhase et al. 2006).

Daily et al. (1998) disclose a significant reduction in the recruitmentof macrophages into distal degenerating nerve in complement-depletedanimals. Complement depletion also decreased macrophage activation, asindicated by their failure to become large and multivacuolated and theirreduced capacity to clear myelin. In the normal situation the myelin iscleared, the proximal part of the nerve forms sprouts which slowly growalong the path of the degenerated nerve. However, regeneration is slow(2-2.5 mm/day) and the environment of a degenerated nerve is full offactors which inhibit the growth of the axon and the necessary growthfactors can be limiting or even absent. Myelin itself has been proposedto be a major inhibiting factor. Therefore rapid clearance of myelin isconsidered a conditio sine qua non for axonal regeneration. Thus thedelayed clearance of myelin in complement-depleted animals is expectedto result in impaired axonal regeneration. These findings indicate arole for serum complement in both the recruitment and activation ofmacrophages during peripheral nerve degeneration as well as an activerole for macrophages in promoting axonal regeneration.

Indeed U.S. Pat. No. 6,267,955 discloses the methods wherein mononuclearphagocytes are administered at or near a site of injury or disease ofthe central or peripheral nervous system of a mammal in order to effectremoval of the myelin debris that reportedly inhibits axonalregeneration, and for release of macrophage-derived cytokines thatpromote modulation of astrocytes and oligodendrocytes so as to supportaxonal regeneration.

Axonal degeneration is the main cause of disability both in hereditaryand in acquired demyelinating neuropathies. While most currenttherapeutic research aims at restoring myelination, the presentinventors focus on the consequence of demyelination: secondary axonaldegeneration. As a model we have used acute demyelination and axonaldegeneration after crush injury and subsequent regeneration of thenerve. It is an object of the present invention to provide for means andmethods that promote and improve regeneration of nerves.

DESCRIPTION OF THE INVENTION

In the Examples herein we have observed activation of the complement(C)-system in the rat during WD and in human nerve biopsies of chronicdemyelinating neuropathies. The present invention is based on thesurprising finding that axonal regeneration is enhanced in rats that aredeficient in the complement C6 factor. This surprising finding opens newways to promote axonal regeneration by manipulation of the complementsystem and/or macrophage activation.

In a first aspect, therefore, the invention pertains to a method fortreating a condition requiring axonal regeneration. The method comprisesthe administration of an inhibitor of a mammalian complement system, orthe administration of a medicament (e.g. a pharmaceutical composition)comprising the inhibitor. Preferably an effective amount of theinhibitor is administered. Thus, in this aspect the invention pertainsto an inhibitor of a mammalian complement system, or a medicamentcomprising the inhibitor, for use in a method for treating a conditionrequiring axonal regeneration. Similarly, in this aspect the inventionpertains to the use of an inhibitor of a mammalian complement system forthe manufacture of a medicament for the treatment of a conditionrequiring axonal regeneration. In the methods and uses of the inventionthe medicament preferably is a medicament for facilitation of axonalregeneration.

In the context of the present invention “facilitating axonalregeneration” is distinguished from reducing or preventing axonaldegeneration. Facilitation (or promotion) of axonal regeneration isherein understood to mean that regeneration of an axon is improved insubjects that are treated as compared to non-treated subjects. Improvedregeneration of an axon preferably is regeneration that occurs at anearlier point in time (after axonal injury or after start of thetreatment) in treated subject as compared to non-treated subjects.Improved regeneration of an axon may also comprise regeneration thatoccurs at a higher rate and/or to a larger extent in treated subject ascompared to non-treated subjects. A medicament according to theinvention thus preferably produces a gain of sensory or motor function.

Improvement in axonal regeneration is preferably determined byfunctional tests that are relatively easily conducted in human subjects,e.g. recovery of sensory or motor function is preferably determined in astandardised test as is available in the art (see e.i. Wong et al.,2006; Jerosch-Herold, 2005). Suitable tests preferably are quantitative,standardised and more preferably have had their psychometric propertiesevaluated and quantified. Such tests include e.g. the Weinstein EnhancedSensory Test (WEST) or the Semmes-Weinstein Monofilament Test (SWMT) andthe shape-texture identification (STI) test for tactile gnosis. Improvedaxonal regeneration may experimentally be determined in test animals byfunctional tests for recovery of sensory or motor function as describedby Hare et al. (1992) and De Koning et al. (1986). A medicamentaccording to the invention thus preferably produces a gain of sensory ormotor function, as may be determined in e.g. an above-indicated test.

Improved axonal regeneration may also be experimentally determined intest animals by histological examination. E.g. improved remyelinationmay be determined by comparing measurements of myelin sheaths around theaxon in treated animals vs. non-treated animals, whereby a thickermyelin sheath is indicative of improved remyelination. More efficientaxonal regeneration may be determined as the production of single, largediameter, axon sprouts in treated animals as compared to clusters ofsmaller axons in non-treated animals.

The appropriate dose of the inhibitor is that amount effective topromote axonal regeneration as may be seen by improvement of sensory ormotor function as described above. By “effective amount,” “therapeuticamount,” or “effective dose” is meant that amount sufficient to elicitthe desired pharmacological or therapeutic effects, thus resulting ineffective treatment of the injury or disorder.

In order to minimise nerve injury and/or to facilitate axonalregeneration at soon as possible, in the methods of the invention, themedicament is preferably administered shortly after the occurrence ofthe nerve injury, i.e. within 24, 12, 6, 3, 2, or 1 hours, morepreferably within 45, 30, 20 or 10 minutes after the occurrence of thenerve injury. In one embodiment of the invention, the medicament may beadministered (e.g. as a precautionary measure) prior to surgery with arisk of nerve injury (see below), so as to minimise nerve injury and/orto facilitate axonal regeneration immediately upon surgical injury ofthe nerve.

Conditions Requiring Axonal Regeneration

A variety of conditions that require axonal regeneration may be treatedwith the methods and/or the medicaments of the invention. The conditionsinclude injury of the PNS as well as injury of the CNS. The conditionsinclude nerve trauma as a result of physical injuries as well asresulting from disease. Such diseases include immune-mediatedinflammatory disorders or injuries and/or progressive neurodegenerativedisorders which may be acquired and/or hereditary.

The physical injuries of the PNS and CNS may be traumatic injuries,including surgical injuries, or non-traumatic injuries. Traumatic PNSand CNS injuries that may be treated with the methods and/or themedicaments of the invention include spinal cord lesions as well astraumatic wounds to peripheral nerves, including injuries fromcollisions, motor vehicle accidents, gun wounds, fractures,dislocations, lacerations, or some other form of penetrating trauma.Peripheral nerves injured through trauma that may be treated include thedigital, median, ulnar, radial, facial, spinal accessory and brachialplexus nerves.

Surgical PNS injuries are herein understood as injuries to peripheralnerves that arise when it becomes clinically necessary to remove ordissect a nerve during a surgical procedure. This occurs in thousands ofsurgical procedures each year. One example of surgically injuredperipheral nerves that may be treated with the methods and/ormedicaments of the invention include e.g. the cavernous nerves thatsupport erectile function and bladder control; these nerves are oftendamaged during surgical removal of a prostate tumour and the tissuearound it. Another example of a surgically injured peripheral nerve thatmay be treated in accordance with the invention is the phrenic nerveafter coronary artery bypass grafting (CABG).

Non-traumatic physical PNS injuries that may be treated with the methodsand/or the medicaments of the invention include compression and/oradhesion of peripheral nerves, also known as entrapment syndromes. Themost common entrapment syndrome is carpal tunnel syndrome.

In addition immune-mediated inflammatory disorders or injuries may betreated with the methods and/or the medicaments of the invention. Theseinclude demyelinating diseases of the central and peripheral nervoussystems that are believed to have an autoimmune basis and result innerve demyelination as a result of damage caused to oligodendrocytes orto myelin directly. Such demyelinating diseases include e.g.Guillain-Barré syndrome (GBS; also referred to as inflammatorydemyelinating polyneuropathy, acute idiopathic polyradiculoneuritis,acute idiopathic polyneuritis, French Polio and Landry's ascendingparalysis). Preferably, methods and/or the medicaments of the inventionare applied to promote axonal regeneration subsequent to acute phase inGBS. Similarly chronic inflammatory demyelinating polyneuropathy (CIDP),considered the chronic counterpart of GBS, may be treated with themethods and/or the medicaments of the invention.

Multiple sclerosis (MS) is another demyelinating disease that may betreated with the methods and/or the medicaments of the invention.

Further neurodegenerative CNS and/or PNS disorders with a geneticcomponent that may be treated with the methods and/or the medicaments ofthe invention include Amyotrophic Lateral Sclerosis (ALS, sometimescalled Lou Gehrig's disease), Charcot-Marie-Tooth disease (HereditaryMotor and Sensory Neuropathy, HMSN) and Huntington Disease (HD).

The Complement System

The complement system (see McAleer and Sim, 1993; Reid and Law, 1988) isconcerned with host defence against infection. Upon activation of thesystem a catalytic set of reactions and interactions occur resulting inthe targeting of the activating cell, organism or particle fordestruction. The complement system comprises a set of over 30 plasma andmembrane proteins that act together in a regulated cascade system toattack extra cellular forms of pathogens (e.g., bacterium). Thecomplement system includes two distinct enzymatic activation cascades,the classical and alternative pathways which converge in a commonterminal non-enzymatic pathway known as the membrane attack pathway.

The first enzymatically activated cascade, known as the classicalpathway, comprises several components, C1, C4, C2, C3 and C5 (listed byorder in the pathway). Initiation of the classical pathway of thecomplement system occurs following binding and activation of the firstcomplement component (C1) by both immune and non-immune activators. C1comprises a calcium-dependent complex of components C1q, C1r and C1s,and is activated through binding of the C1q component. C1q contains sixidentical subunits and each subunit comprises three chains (the A, B andC chains). Each chain has a globular head region that is connected to acollagen-like tail. Binding and activation of C1q by antigen-antibodycomplexes occurs through the C1q head group region. Numerousnon-antibody C1q activators, including proteins, lipids and nucleicacids, bind and activate C1q through a distinct site on thecollagen-like stalk region. The C1qrs complex then catalyzes theactivation of complement components C4 and C2, forming the C4b2a complexwhich functions as a C3 convertase.

The second enzymatically activated cascade, known as the alternativepathway, is a rapid, antibody-independent route for complement systemactivation and amplification. The alternative pathway comprises severalcomponents, C3, Factor B, and Factor D (listed by order in the pathway).Activation of the alternative pathway occurs when C3b, a proteolyticallycleaved form of C3, is bound to an activating surface agent such as abacterium. Factor B is then bound to C3b, and cleaved by Factor D toyield the active enzyme, Ba. The enzyme Ba then cleaves more C3 togenerate more C3b, producing extensive deposition of C3b-Ba complexes onthe activating surface.

Thus, both the classical and alternate complement pathways produce C3convertases that split factor C3 into C3a and C3b. At this point, bothC3 convertases further assemble into C5 convertases (C4b2a3b andC3b3bBb). These complexes subsequently cleave complement component C5into two components: the C5a polypeptide (9 kDa) and the C5b polypeptide(170 kDa). The C5a polypeptide binds to a 7 transmembrane G-proteincoupled receptor, which was originally associated with leukocytes and isnow known to be expressed on a variety of tissues including hepatocytesand neurons. The C5a molecule is the primary chemotactic component ofthe human complement system and can trigger a variety of biologicalresponses including leukocyte chemotaxis, smooth muscle contraction,activation of intracellular signal transduction pathways,neutrophil-endothelial adhesion, cytokine and lipid mediator release andoxidant formation.

The larger C5b fragment binds sequentially to later components of thecomplement cascade, C6, C7, C8 and C9 to form the C5b-9 membrane attackcomplex (“MAC”). The lipophylic C5b-9 MAC can directly lyseerythrocytes, and in greater quantities it is lytic for leukocytes anddamaging to tissues such as muscle, epithelial and endothelial cells. Insublytic amounts, the C5b-9 MAC can stimulate upregulation of adhesionmolecules, intracellular calcium increase and cytokine release. Inaddition, at sublytic concentrations the C5b-9 MAC can stimulate cellssuch as endothelial cells and platelets without causing cell lysis. Thenon-lytic effects of C5a and the C5b-9 MAC are comparable andinterchangeable.

Although the complement system has an important role in the maintenanceof health, it has the potential to cause or contribute to disease.

Inhibitors of the Complement System

An inhibitor of a mammalian complement system for use in the methodsand/or medicaments of the present invention may be an antagonist,polypeptide, peptide, antibody, anti-sense oligonucleotide, aptamer,miRNA, ribozyme, siRNA, or small molecule. The inhibitor preferablyinhibits or blocks the formation of the membrane attack complex. Theinhibitor preferably blocks activation of the complement system throughboth the classical and alternative pathway of complement. A preferredinhibitor is an inhibitor that blocks C3 convertase and MAC assembly. Afurther preferred inhibitor is an inhibitor that blocks one or more ofC5, C6, C7, C8 and C9. The following compounds may thus be used in themethods and/or medicaments of the invention.

A preferred complement inhibitor for use in the present invention is acomplement regulator, complement receptor or derivatives thereof. Theseinclude all natural regulators of the complement system such asC1-inhibitor, CR1, DAF, MCP, and CD59. Further included are derivativesof natural regulators of the complement system containing commonstructural units (CSR). CR1, MCP, DAF, C4 bp, fH all contain shortconsensus repeats (SCR). The SCR is a structural motif of 60-70 aminoacids that is tandemly repeated 30 times in the F-allotype of CR1; thenumber of repeats can vary between allotypes. The consensus sequence ofthe SCR includes 4 cysteines, a glycine and a tryptophan that areinvariant among all SCR. Sixteen other positions are conserved, with thesame amino acid or a conservative replacement being found in over halfof the 30 SCRs (Klickstein, et al., 1987, 1988; Hourcade, et al., 1988).Preferably the complement regulator containing SCRs comprises at least3, 6, 12, 25 or 30 SCRs. Preferably the complement regulator containingSCRs is a soluble derivative of a complement receptor. Suitable examplesthereof include e.g. sCR1 (TP10) which contains 30 SCRs, sMCP, sDAF, andCAB-2, which is a DAF/MCP hybrid. Modifications of these molecules allowtargeting to membranes.

Soluble CR1 is a preferred inhibitor of complement activation becauseonly CR1 combines specificity for both C3b and C4b with capabilities fordissociating the C3 convertases of both pathways and for cofactoractivity in the proteolytic inactivation of C3b and C4b by factor I. Inaddition, these functions of CR1 are not restricted by alternativepathway activating functions, making the receptor suitable forsuppressing activation by non-immunologic stimuli and inhibition of bothclassical and alternative pathway complement activation. Soluble CR1(sCR1) fragments have been prepared by recombinant DNA techniques, usingcDNA lacking the transmembrane and cytoplasmic domains (WO 89/09220; WO91/05047). Preferred sCR1 molecules for use in the methods and/ormedicaments of the invention are 1) a soluble CR1 protein that has thecharacteristics of the protein expressed by a Chinese hamster ovary cellDUX B11 carrying plasmid pBSCR1/pTCSgpt as deposited with the ATCC andassigned accession number CRL 10052; or 2) soluble complement receptor 1TP10 (Avant Immunotherapeutics, Inc.).

A further complement regulator for use in the methods and/or medicamentsof the invention is C1-inhibitor (C1INH). C1INH is a member of the serinprotease inhibitor (serpins) family and it binds to the active site onboth C1r and C1s inhibiting formation of the C1 complex. An advantage ofplasma derived C1INH is its a long serum half-life (70 hours) in humans.Alternatively transgenic human C1INH may be used (WO 01/57079).

Yet another membrane-bound complement receptor for use in the methodsand/or medicaments of the invention is Crry-Ig (Quigg et al., 1998).Crry is a membrane complement inhibitor with decay accelerating activityat the 3 convertase level, inhibiting both the classical and alternativepathway of complement. It also possesses cofactor activity comparable tothat of CR1 for the factor I-mediated cleavage of C3b and C4b. Crry-Igis a recombinant, soluble protein with an increased half-life (40 hours)due to fusion of Crry with the Fc portion of a non-complement-activatingmouse IgG1 partner. Overall, Crry-Ig is potent complement inhibitor.

Antibodies or antibody-fragments against complement components are afurther class of compounds that are of use in the methods and/ormedicaments of the invention. In principle antibodies against anycomplement factor may be of use. However, preferred antibodies areantibodies that block C3 convertase and/or MAC assembly. A furtherpreferred antibody is an antibody that blocks one or more of C5, C6, C7,C8 and C9. Preferably the antibody or fragment thereof is a monoclonalantibody (MAb). MAbs to complement components can be prepared using awide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof. For example, monoclonal antibodies can be produced usinghybridoma techniques including those known in the art and taught (i.e.Harlow et al., 1998; Hammerling, et al., 1981).

For treating humans, the anti-complement MAbs would preferably be usedas chimeric, deimmunised, humanised or human antibodies. Such antibodiescan reduce immunogenicity and thus avoid human anti-mouse antibody(HAMA) response. It is preferable that the antibody be IgG4, IgG2, orother genetically mutated IgG or IgM which does not augmentantibody-dependent cellular cytotoxicity (Canfield and Morrison, 1991)and complement mediated cytolysis (Xu et al., 1994; Pulito et al.,1996). Chimeric antibodies are produced by recombinant processes wellknown in the art, and have an animal variable region and a humanconstant region. Humanised antibodies have a greater degree of humanpeptide sequences than do chimeric antibodies. In a humanised antibody,only the complementarity determining regions (CDRs) which areresponsible for antigen binding and specificity are animal derived andhave an amino acid sequence corresponding to the animal antibody, andsubstantially all of the remaining portions of the molecule (except, insome cases, small portions of the framework regions within the variableregion) are human derived and correspond in amino acid sequence to ahuman antibody. See Riechmann et al., 1988; Winter, U.S. Pat. No.5,225,539; Queen et al., U.S. Pat. No. 5,530,101. Deimmunised antibodiesare antibodies in which the T and B cell epitopes have been eliminated,as described in WO9852976. They have reduced immunogenicity when appliedin vivo.

Human antibodies can be made by several different ways, including by useof human immunoglobulin expression libraries (Stratagene Corp., LaJolla, Calif.) to produce fragments of human antibodies (VH, VL, Fv, Fd,Fab, or (Fab′)2, and using these fragments to construct whole humanantibodies using techniques similar to those for producing chimericantibodies. Human antibodies can also be produced in transgenic micewith a human immunoglobulin genome. Such mice are available fromAbgenix, Inc., Fremont, Calif., and Medarex, Inc., Annandale, N.J.

One can also create single peptide chain binding molecules in which theheavy and light chain Fv regions are connected. Single chain antibodies(“ScFv”) and the method of their construction are described in U.S. Pat.No. 4,946,778. Alternatively, Fab can be constructed and expressed bysimilar means (Evans et al., 1995).

Another class of antibodies that may be used in the context of thepresent invention are heavy chain antibodies and derivatives thereof.Such single-chain heavy chain antibodies naturally occur in e.g.Camelidae and their isolated variable domains are generally referred toas “VHH domains” or “nanobodies”. Methods for obtaining heavy chainantibodies and the variable domains are inter alia provided in thefollowing references: WO 94/04678, WO 95/04079, WO 96/34103, WO94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/65057, WO01/40310, WO 01/44301, EP 1134231, WO 02/48193, WO 97/49805, WO01/21817, WO 03/035694, WO 03/054016, WO 03/055527, WO 03/050531, WO01/90190, WO 03/025020, WO 04/041867, WO 04/041862, WO04/041865, WO04/041863, WO 04/062551.

All of the wholly and partially human antibodies are less immunogenicthan wholly murine MAbs, and the fragments and single chain antibodiesare also less immunogenic. All these types of antibodies are thereforeless likely to evoke an immune or allergic response. Consequently, theyare better suited for in vivo administration in humans than whollyanimal antibodies, especially when repeated or long-term administrationis necessary. In addition, the smaller size of the antibody fragment mayhelp improve tissue bioavailability, which may be critical for betterdose accumulation in acute disease indications, such as tumourtreatment.

Suitable anti-complement antibodies are already available. E.g. theanti-05 MAb produced by hybridoma 5G1.1 having ATCC designation HB-11625(N19-8) as described in U.S. Pat. No. 6,355,245; anti-C3a MAb fromQuidel, San Diego, Calif. [catalog no. A203,]; anti-human C3aRantibodies hC3aRZ1 and hC3aRz2, as described in Kacani et al., (2001);mouse anti-human C5a antibodies from Hycult Biotechnology BV of theNetherlands [clones 557, 2942 and 2952]; anti-human C5a antibody fromTanox, Inc. [137-26], as described in Fung et al. (2003); C5a antibodiesdisclosed in U.S. Pat. No. 5,480,974; anti-EX1 human C5aR MAb S5/1, asdescribed in Oppermann et al., (1993); anti-05aR MAb S5/1, as describedin Kacani et al., (2001); and anti-05a MAb as described in U.S. Pat. No.5,177,190.

A further compound that may be used in the methods and/or medicaments ofthe invention is cobra venom factor (CVF) or a derivative thereof thatdepletes C3 by binding Factor B and formation of a C3 convertaseactivity that is resistant to natural fluid phase regulators. Apreferred CVF derivative is e.g. a derivative that can be targeted tothe site of injury.

Other useful compounds include poly-anionic inhibitors of complementsuch as heparin, N-acetylated heparin and suramin. Heparin inhibits C bybinding to C1, blocking C3 convertase and MAC assembly. N-acetylatedheparin has a reduced anticoagulant activity.

In addition a variety of synthetic and/or natural small molecules thatinhibit complement may be used in the methods and/or medicaments of theinvention, e.g. the natural inhibitors K-76COOH (derived fromStachybotrys complements), which inhibits C5, and rosmaric acid derivedfrom Rosemary, which binds and inhibits C3b and thereby preventsconvertase formation, synthetic protease inhibitor s such as e.g.nafamastat mesilate (FUT-175), which binds Cir, C1s, Factor D and C3/C5convertase, inhibitors of the C1 complex such e.g. C1s-INH-248 andBCX-1470 (already tested in humans for safety), peptide inhibitors suchas molecules containing parts of or derived from complement bindingnatural molecules, such as e.g. derivatives containing thecarboxyterminal part of serpins, compstatin (a 13 a.a. cyclic moleculethat binds C3), and the C5 receptor agonists PMX53 and PMX205.

Methods for producing nucleic acid inhibitors of complement such asanti-sense oligonucleotide, aptamer, miRNA, ribozyme, siRNA, are knownto the skilled person persé. Preferably such nucleic acid inhibitorscomprise one or more modified nucleotides such as e.g. 2′-O substitutedribonucleotides, including alkyl and methoxy ethyl substitutions,peptide nucleic acid (PNA), locked nucleic acid (LNA) and morpholinoantisense oligonucleotides and ethylene-bridged nucleotides (ENA) andcombinations thereof.

In the above methods of the invention, the compounds may be administeredby any convenient route, for example by infusion or bolus injection.Various delivery systems are known and can be used for delivery of theinhibitor compounds. These include encapsulation in liposomes,microparticles, or microcapsules. Although in the methods of theinvention administration of the compounds by oral and/or mucosal routes(intranasal, inhalation, rectal) is not excluded, usually the complementinhibitors will be administered parenterally, including e.g.intradermal, intramuscular, intraperitoneal, intravenous, andsubcutaneous routes. The compounds may be administered systemically ormay be used by local, topical or regional administration at or near asite of disease or injury, e.g. using injection and/or anyneurosurgically suitable technique.

The invention further relates to a pharmaceutical preparation comprisingas active ingredient a complement inhibitor as defined above. Thecomposition preferably at least comprises a pharmaceutically acceptablecarrier in addition to the active ingredient. The pharmaceutical carriercan be any compatible, non-toxic substance suitable to deliver theinhibitors to the patient. Sterile water, alcohol, fats, waxes, andinert solids may be used as the carrier. Pharmaceutically acceptableadjuvants, buffering agents, dispersing agents, and the like, may alsobe incorporated into the pharmaceutical compositions.

For oral administration, the inhibitor can be administered in soliddosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. Activecomponent(s) can be encapsulated in gelatine capsules together withinactive ingredients and powdered carriers, such as glucose, lactose,sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonateand the like. Both tablets and capsules can be manufactured as sustainedrelease products to provide for continuous release of medication over aperiod of hours. Compressed tablets can be sugar coated or film coatedto mask any unpleasant taste and protect the tablet from the atmosphere,or enteric-coated for selective disintegration in the gastrointestinaltract. Liquid dosage forms for oral administration can contain colouringand flavouring to increase patient acceptance.

The inhibitors are however preferably administered parentally. Suitablecarriers for parental formulations include saline, buffered saline,dextrose, and water. Typically compositions for parenteraladministration are solutions in sterile isotonic aqueous buffer.Sterilisation is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilisation andreconstitution. A typical composition for intravenous infusion could bemade up to contain 10 to 50 ml of sterile 0.9% NaCl or 5% glucoseoptionally supplemented with a 20% albumin solution and an appropriateamount (1 to 1000 μg) of the inhibitor. A typical pharmaceuticalcomposition for intramuscular injection would be made up to contain, forexample, 1-10 ml of sterile buffered water and 1 to 1000 μg of the ofthe inhibitor. Methods for preparing parenterally administrablecompositions are well known in the art and described in more detail invarious sources, including, for example, Remington's PharmaceuticalScience (15th ed., Mack Publishing, Easton, Pa., 1980) (incorporated byreference in its entirety for all purposes). Where necessary, thecomposition may also include a solubilising agent and a localanaesthetic such as lignocaine to ease pain at the site of theinjection. Generally, the ingredients will be supplied either separatelyor mixed together in unit dosage form, contained in a hermeticallysealed container such as an ampoule or sachette indicating the quantityof active agent in activity units. Where the composition is to beadministered by infusion, it can be dispensed with an infusion bottlecontaining sterile pharmaceutical grade ‘Water for Injection’ or saline.Where the composition is to be administered by injection, an ampoule ofsterile water for injection or saline may be provided so that theingredients may be mixed prior to administration.

In those methods where the inhibitor is a polypeptide or antibody it maybe purified from mammalian, insect or microbial cell cultures, from milkof transgenic mammals or other source and be administered in purifiedform together with a pharmaceutical carrier as a pharmaceuticalcomposition. Methods of producing pharmaceutical compositions comprisingpolypeptides are described in U.S. Pat. Nos. 5,789,543 and 6,207,718.The preferred form depends on the intended mode of administration andtherapeutic application. The concentration of the polypeptides orantibodies of the invention in the pharmaceutical composition can varywidely, i.e., from less than about 0.1% by weight, usually being atleast about 1% by weight to as much as 20% by weight or more.

In this document and in its claims, the verb “to comprise” and itsconjugations is used in its non-limiting sense to mean that itemsfollowing the word are included, but items not specifically mentionedare not excluded. In addition, reference to an element by the indefinitearticle “a” or “an” does not exclude the possibility that more than oneof the element is present, unless the context clearly requires thatthere be one and only one of the elements. The indefinite article “a” or“an” thus usually means “at least one”.

DESCRIPTION OF THE FIGURES

FIG. 1. The effect of complement C6-deficiency on regeneration of thetibial nerve. The right sciatic nerve was crushed for 30 seconds in wildtype and C6-deficient PVG rats. Tibial nerve was analyzed 1 and 5 weeksafter injury. Control picture: left tibial nerve of a PVG rat.

FIG. 2. C6 deficiency leads to a delayed influx/activation of phagocyticcells. ED1 (CD68) immunoreactive (-ir) cells were counted innon-consecutive sections of sciatic nerves from WT (wild type), C6−/−(C6-deficient) and C6+ (C6-deficient rats supplemented with C6) rats at0, 24, 48 and 72 h post-injury. Statistical significance indicated bythe asterisk (*) refers to p<0.05.

FIG. 3. The effect of C6 reconstitution on regeneration. Analysis ofmyelinated axons during regeneration. Light microscopy on semi-thinsections of the proximal site of the rat tibial nerve at 5 wkpost-injury. From left to right: uncrushed nerve; wild type nerve (WT);C6-deficient nerve (C6−/−); and, C6-deficient nerve reconstituted withC6 (C6+).

FIG. 4. The effect of C6 reconstitution on functional recovery. Recoveryof sensory function as measured with the footflick apparatus at currentsranging from 0.1 mA to 0.5 mA. Values are normalised to control levels.The arrow (→) indicates the time at which the crush injury wasperformed. WT=wild type rats; C6−/−=C6-deficient rats; and, C6+=C6reconstitution in C6-deficient animals. Statistical significance betweenC6−/− and WT (*) or C6+ (†) is for p<0.05.

FIG. 5. Recombinant human C1-inhibitor (rhC1INH) inhibits complementactivation after crush. C1q, C4c and C3c immunostaining of injured wildtype rat sciatic nerves treated with rhC1INH or vehicle (PBS) alone.

FIG. 6. The effect of soluble CR1 on post traumatic nerve regeneration.Recovery of sensory function as measured with the footflick apparatus atcurrents ranging from 0.1 mA to 0.5 mA. Values are normalised to controllevels. The arrow (→) indicates the time at which the crush injury wasperformed. PBS=control with vehicle only; sCR1=soluble CR1.

FIG. 7. Activation of macrophages after nerve crush is dependent onactivation of a downstream component of the complement cascade. CD68positive cells were determined by immunostaining with the ED1 antibody.In wild type (WT) and vehicle (PBS) treated animals the number of CD68positive cells in the distal part of the lesioned sciatic nerveincreased 72 hours after nerve crush. Treatment with sCR1 blocked thisactivation to a similar level as seen in C6 deficient rats (C6−). C6reconstitution in the C6 deficient animals (C6+) resulted in almostcomplete recovery from this block in activation.

FIG. 8. Recovery of function. (a) Sciatic Functional Index (SFI) andfootprints after sciatic nerve crush injury (time=0) in wildtype (n=8),C6^(−/−) (n=8) and C6⁺ (n=8) rats showing recovery of motor functionover a period of 5 weeks post-injury. Control levels are near 0 whereasvalues near −140 indicate complete loss of function (week 1). Theasterisks (*) refers to significant differences between the wildtype andC6^(−/−) group of rats whereas the cross(†) refers to significantdifferences between C6^(−/−) and C6⁺ group of rats with p≦0.05determined by two way ANOVA test with Bonferroni correction. The widertoe spread in the C6^(−/−) footprint (week 4) compared to the wildtypeand C6⁺ footprints indicates increased muscle strength. (b) Footflickanalysis after sciatic nerve crush injury (time=0) in wildtype (n=8),C6^(−/−) (n=8) and C6⁺ (n=8) rats showing recovery of sensory functionover a period of 5 weeks post-injury. Values are expressed as percentageof control levels (100% function). The asterisks (*) refers tosignificant differences between the wildtype and C6^(−/−) group of ratswhereas the cross(†) refers to significant differences between C6^(−/−)and C6+ group of rats with p≦0.05 determined by two way ANOVA test withBonferroni correction.

FIG. 9. Recovery of sensory function of C inhibited rats. Footflickanalysis after sciatic nerve crush injury (time=0) in wildtype PBS-(n=6) and sCR1- (n=6) treated rats showing recovery of sensory functionover a period of 5 weeks post-injury. Values are expressed as percentageof control levels (100% function). The asterisks (*) refers p≦0.05determined by two way ANOVA test with Bonferroni correction.

FIG. 10. Pathology. Thionine staining and electron microscopy ofcross-sections of the distal ends of tibial nerves from uninjured (n=6),wildtype (n=5), C6^(−/−) (n=5) and sCR1-treated (n=6) rats at 5 weeksfollowing the crush injury. Note the presence of regenerative clustersof small caliber, thinly myelinated axons in the wildtype nerve (arrows→) whereas single large caliber axons are present in the C67 andsCR1-treated nerves, similarly to the uninjured control. Bar is 50 μm(light microscopy, left panels) and 10 μm (electron microscopy, rightpanels).

FIG. 11 a and 11 b. sCR1 inhibition of complement activation (A) PlasmasCR1 levels in sCR1-treated rats, showing concentration of sCR1 overtime with daily treatment. (B) Plasma hemolytic activity of PBS- andsCR1-treated rats, showing decreased activity in the sCR1-treated ratscompared to the PBS-treated controls. (A, B) Day 0 is the day of thecrush injury. Rats received i.p injections of sCR1 (15 mg/kg/day) or PBS(equal volume) at days (−1, 0, 1, 2, 3, 4, 5 and 6). Blood was collectedimmediately before each treatment. Data represents mean±SD. Statisticalsignificance is determined by two-way ANOVA with Bonferroni correction(* is p≦0.001).

FIG. 12. sCR1 inhibition of complement activation. Quantification of MACimmunoreactivity expressed as percentage of total area scored. Datarepresents mean±SD. Statistical significance is determined by two-wayANOVA with Bonferroni correction (* is p≦0.001).

FIG. 13. Analysis of macrophages. Quantification of CD68-ir cells innon-consecutive sections of sciatic nerves, showing a high number ofcells in the PBS-treated nerves and slight increase in the sCR1-treatednerves, compared to the uninjured nerve. Data represents mean±SD.Statistical significance is determined by two-way ANOVA with Bonferronicorrection.

FIG. 14. Analysis of macrophages. Size distribution of CD68-ir cellssciatic nerves from uninjured nerve (a), PBS-treated (b) andsCR1-treated (c) nerves at 3 days post-injury. Note the shift in thepeak of CD68-ir cell size distribution from a size of 0-40 μm² in theuninjured and sCR1-treated nerves to a size of 40-120 μm² in thePBS-treated nerves.

FIG. 15. Analysis of alternative pathway activation. (a) Westernblotting analysis of rat sciatic nerves at 2 days post-injury, showinghigher amount of cleaved fBb protein in the injured nerves compared tothe uninjured controls. (b) Relative quantification of fBbimmunoreactive bands. The fBb immunoreactivity in uninjured controls isdefined as 1.0 fold relative expression. Values are normalized to totalprotein load and represent mean±SD of three blots. Statisticalsignificance is determined by unpaired t-test.

EXAMPLES 1. Example 1 Improved Post-Traumatic Nerve Recovery inComplement Component C6 Deficient Rats as Compared to Wild-Type Rats 1.1Electron Microscopic Analysis

We have explored the role of the complement system in acute and chronicnerve injury and during regeneration. As a model we used the complementC6 deficient PVG rat strain (Bhole and Stahl, 2004) and compared thiswith wild type PVG rats. Since the complement system has many functionswe chose an animal model in which only the most terminal effectors ofthe complement cascade was defective.

The effect of complement inhibition on nerve regeneration was studied inthe acute model of nerve crush (Glass, 2004). The right sciatic nervewas crushed for 30 seconds in wild type as well as in the C6-deficientPVG rats. Tibial nerve was then analyzed 1 and 5 weeks after injury (seeFIG. 1).

At one week, electron microscopy shows equally severe degeneration inwild type and in C6 deficient rats. At 5 weeks, the C6 deficient ratsalready show myelinated axons, whereas the wild type rats exhibitincipient recovery. In the C6 deficient animals most myelinated axonsshow the normal one to one ratio with Schwann cells (compare with thecontrol picture of the left tibial nerve of a PVG rat). In contrast, inthe wild type rats there are several myelinated fibres in eachregenerative cluster.

We found two effects of C6 deficiency on post-traumatic nerve recovery:

1) The clearance of myelin during Wallerian degeneration was delayed inthe C6 deficient animals. Wild type rats showed signs of WD (myelindegeneration, macrophage activation) already after 24 hours. InC6-deficient animals this process was delayed. Only after 72 hrs myelindegeneration was visible and macrophage activation did not occur. Afterone week both types of animals showed severe nerve degeneration.

2) Unexpectedly however, the post-traumatic nerve recovery was muchbetter in complement component C6 deficient rats compared to wild-typerat. Remyelination of single axons occurred much faster in the C6deficient animals and the sprouting process was more efficient since asingle, large diameter, axon sprout was produced rather than a clusterof smaller axons. See FIG. 1.

1.2 C6 Deficiency Leads to a Delayed Influx/Activation of PhagocyticCells

In view of the important role of macrophages in myelin clearance, wenext analyzed the number and activation state of macrophages aftercrush.

ED1 (CD68) immunoreactive (-ir) cells were counted in non-consecutivesections of crushed sciatic nerves at 0, 24, 48 and 72 hr post-injurythat were taken from wild type rats, C6 deficient rats and C6 deficientrats that were supplemented with C6, respectively.

In both the wild type and C6 deficient animals, CD68 (ED1 antibody)positive cells accumulated in the crushed nerve. However, the C6deficient animals showed a delayed appearance of CD68 positive cells(FIG. 2, compare solid and dotted line). C6 suppletion restored theaccumulation of CD68 cells (see 72 hr time point). In the C6 deficientanimals there was a lack of activation of macrophages, as assayed byimmunohistochemistry CR3 (ED7 antibody) staining (not shown).

Since lymphnodes of these animals contain CR3 positive cells we couldexclude that the C6 deficient animals are defective in macrophageactivation per se. In addition, upon C6 reconstitution, the accumulationof CD68 positive cells and CR3 expression on macrophages was restoredand subsequently myelin degeneration occurred. This directly links stepsin the complement pathway downstream of C6, i.e. Membrane Attack Complex(MAC) formation to WD.

After 7 days equal number of CD68 positive cells were found in the C6deficient and wild type cells. These cells do not display ED7 (CR3) inthe C6 deficient animals and are most likely not activated macrophages(data not shown).

1.3 Neuropathological and Functional Assays of C6 Deficiency andReconstitution

FIG. 3 shows light microscopic analysis of myelinated axons duringregeneration. Semi-thin sections of the proximal site of the rat tibialnerve were analysed at 5 weeks post-injury of wild type rats, C6deficient rats and C6 deficient rats reconstituted with C6. Few thinlymyelinated axons are present in the wild type (WT) nerve while manythickly myelinated axons are present in the C6 deficient (C6−/−) nerve.The nerve from the rat that was reconstituted with C6 (C6+) shows lessmyelinated axons than the C6 deficient nerve.

FIG. 4 shows the effect of C6 reconstitution on functional recovery ofthe nerve. Recovery of sensory function was measured with the footflickapparatus at currents ranging from 0.1 mA to 0.5 mA. Values werenormalised to control levels. The arrow (→) indicates the time at whichthe crush injury was performed. Wild type rats take 4 weeks to fullyrecover while C6 deficient rats are already recovered at 3 weekspost-crush. C6 reconstitution in C6 deficient animals results in thewild type (slow) regeneration phenotype after crush. Statisticalsignificance between C6−/− and WT (*) or C6+ (†) is for p<0.05.

We conclude that the observed effect on regeneration of the PNS aftercrush is due to the C6 deficiency since reconstitution of the C6deficient rats with purified human C6 restores the wild type phenotypein neuropathological and functional assays (FIGS. 3 and 4).

Example 2 Inhibition of Complement Activation after Nerve Crush by HumanC1-Inhibitor

We tested whether recombinant human C1-inhibitor (rhC1INH; obtained fromPharming, Leiden, The Netherlands) is able to inhibit the rapid (1 hr)complement activation after nerve crush. FIG. 5 shows C1q, C4c and C3cimmunostaining of injured wild type rat sciatic nerves treated withrhC1INH or vehicle (PBS) alone at 1 hour after nerve crush. Highimmunoreactivity for C1q is present in all crushed nerves, confirmingC1q up-regulation after the crush injury. C4c and C3c immunoreactivitywas detected in the PBS-treated nerves as expected but no C4c and C3cimmunoreactivity was detected in the nerves from the rhC1INH treatedrats sacrificed at 1 hr post-injury. This demonstrates effectiveblockade of the complement cascade by rhC1INH after crush and suggeststhat the alternative pathway of complement activation is not involved inthe crush injury model of Wallerian degeneration. Thus, activation ofthe complement cascade after nerve crush occurs through the classicalpathway. There is one caveat however: due to the short half life ofrhC1INH in rats, we could only monitor C3 and C4 cleavage 1 hr aftercrush. Therefore we cannot exclude that activation through thealternative pathway occurs at a later time point.

Example 3 The Effect of Soluble CR1 on Post Traumatic Nerve Regeneration

Next, we tested the effect of soluble CR1 (sCR1) on post traumatic nerveregeneration. sCR1 inhibits the C3/C5 convertase, and thereby affectsboth the classical and alternative pathway of the complement system.

Wild type PVG rats were treated with soluble CR1 (TP10 from AvantImmunotherapeutics, Inc.) at a dose of 15 mg/kg/day (TP10 soluble CR1was obtained from Prof. P. Morgan, Cardiff, UK). Control rats weretreated with the same volume (600 up of vehicle alone (PBS). Soluble CR1or PBS was delivered i.p. 24 hours before the crush and every followingday for a maximum of 8 injections (up to day 6 after crush). The sciaticnerve of the right leg was crushed and the left leg served as control.Both histology and sensory function were analysed.

FIG. 6 shows that in a functional analysis with the footflick test afaster recovery of the sensory function is seen in the sCR1-treatedanimals compared to the PBS-treated. The footflick test was performed asdescribed above in Example 1.3.

Histological analysis of nerves at 72 hrs after the crush shows thatsCR1 strongly inhibited the influx and activation of macrophages (seeFIG. 7). sCR1 treatment resulted in similar levels of inhibition ofmacrophage activation as measured by CD68 positivity as compared todeficiency of C6.

Example 4 Inhibition of Complement Activation Facilitates AxonRegeneration and Recovery in a Model of Peripheral Nerve Injury 4.1Methods 4.1.1 Animals

This study was approved by the Academic Medical Center Animal EthicsCommittee and complies with the guidelines for the care of experimentalanimals. Male 12 weeks old PVG/c (wildtype) were obtained from Harlan(UK) and PVG/c⁻ (C6^(−/−)) rats were bred in our facility. The animalsweighed between 200 g and 250 g and were allowed to acclimatize for atleast two weeks before the beginning of the study. Animals were kept inthe same animal facility during the entire course of the experiment andmonitored for microbiological status according to the FELASArecommendations. Animals were housed in pairs in plastic cages. Theywere given rat chow and water ad libitum and kept at a room temperatureof 20° C. on a 12 hours: 12 hours light:dark cycle.

4.1.2 Genotyping of PVG/c− (C6^(−/−)) Rats

The C6^(−/−) rats carry a deletion of 31 basepairs (bp) in the C6 gene(Bhole and Stahl, 2004). Genotyping was performed according to Ramagliaet al (2007).

4.1.3 Administration of Human C6 for Reconstitution Studies

C6 was purified from human serum (Mead et al., 2002). It wasadministered i.v. in eight C6^(−/−) rats at a dose of 4 mg/kg/day in PBSone day before the crush injury (day −1) and every day thereafter for 1week (day 0, 1, 2, 3, 4, 5, 6). Eight wildtype and eight C6^(−/−) ratswere treated with equal volume of vehicle (PBS) alone. The C6^(−/−) ratsreconstituted with purified human C6 will be indicated in the text asC6⁺.

4.1.4 Administration of sCR1 for Inhibition Studies

Recombinant soluble complement receptor I (sCR1) was obtained aspreviously described (Piddlesden et al., 1994). sCR1 was administeredi.p. in six rats at a dose of 15 mg/kg/day. Six rats were treated withequal volumes of vehicle (PBS) alone. The treatment was given one daybefore the crush injury (day −1) and every day thereafter for 1 week(day 0, 1, 2, 3, 4, 5, 6).

4.1.5 Hemolytic Assay and ELISA

Blood samples from wildtype PBS-treated, C6^(−/−) PBS-treated, C6⁺ andsCR1-treated rats were collected from the tail vein one day before thecrush injury (day −1) and every following day until 1 week post-injury(day 0, 1, 2, 3, 4, 5, 6, 7). All samples were collected immediatelybefore each injection of treatment. Plasma was separated and stored at−80° C. until used to monitor C6 activity and sCR1 inhibitory effect viastandard complement hemolytic assay (Morgan, 2000). Plasma levels ofsCR1 were measured using ELISA assay as previously described (Mulliganet al., 1992) using serial dilutions assayed in duplicates.

4.1.6 Motor and Sensory Test

All experiments were conducted by the same investigator who was blindedof the genotype and treatment groups. Both motor and sensory tests wereperformed at the same time during the day, every week until 5 weekspost-injury. Recovery of motor function was assessed using astandardized walking track analysis and derived sciatic functional index(SFI) according to Hare et al (1992). Briefly, the rats were allowed towalk across a plexiglas platform while their walking patter was recordedby a camera underneath the platform. An index of the sciatic nervefunction was calculated from the recorded footprints using the ImageProanalysis program (Media Cybernatics, The Netherlands). The print length(PL), toe (1^(st) to 5^(th)) spread (TS) and intermediary toe (2^(nd) to4^(th)) spread (IT) were recorded from the uninjured normal foot (NPL,NTS, NIT) and the contralateral foot on the injured experimental side(EPL, ETS, EIT). The SFI was derived with the formula:−38.3*[(EPL−NPL)/NPL]+109.5*[(ETS−NTS)/NTS]+13.3*[(EIT−NIT)/NIT]. Incase on no print produced by the animals, the standard values of EPL=60mm, ETS=6 mm and EIT=6 mm were used according to De Koning et al (1986).Recovery of sensory function was assessed with the footflick testaccording to De Koning et al (1986). Briefly, a shock source with avariable current of 0.1-0.5 mA was used. Recordings were performed oneday before the injury and every week until 5 weeks post-injury. The ratswere immobilized and two stimulation electrodes were placed at the samepoint on the rat foot sole for every animal and stimulation. A responsewas scored positive if the rat retracted its paw. The current (mA) atwhich the retraction occurred was recorded. Values are expressed aspercentage of normal function.

4.1.7 Nerve Crush Injury

All the surgical procedures were performed aseptically under deepisoflurane anesthesia (2.5% vol isoflurane, 1 L/min O₂ and 1 L/min N₂O).The left thigh was shaved and the sciatic nerve was exposed via anincision in the upper thigh. The nerve was crushed for three 10 speriods at the level of the sciatic notch using smooth, curved forceps(No. 7). The crush site was marked by a suture which did not constrictthe nerve. On the right side, sham surgery was performed which exposedthe sciatic nerve but did not disturb it. A suture was also placed. Themuscle and the skin were then closed with stitches. The right leg servedas control. Following the crush, the rats were allowed to recover for 1(wildtype n=5; C6^(−/−) n=5; C6⁺ n=2), 3 (wildtype n=6; C6⁻⁻ n=6; C6⁺n=3) and 5 (wildtype n=5; C6^(−/−) n=5; C6⁺ n=3; wildtype sCR1-treatedn=6; wildtype PBS-treated n=6) weeks.

4.1.8 Tibial Nerve Histology

All animals were intracardially perfused with 4% paraformaldehyde inpiparazine-N—N′-bis(2-ethane sulphonic acid) (PIPES) buffer (pH 7.6),under deep isoflurane anesthesia. Left and right tibial nerves wereremoved from each animal and postfixed with 1% glutaraldehyde, 1%paraformaldehyde and 1% dextran (MW 20,000) in 0.1 M PIPES buffer (pH7.6). They were divided into one proximal and one distal segment of 10mm length. Each segment was conventionally processed into epoxy resin.Semithin resin sections of 0.5 μm were stained with thionine andacridine orange and images were captured with a light microscope (LeicaDM5000B, The Netherlands) connected to a digital camera (Leica DFC500,The Netherlands). Electron microscopy was performed on ultrathinsections of the tibial nerve from wildtype and C6^(−/−) rats at 5 weeksfollowing the crush injury. Sections were contrasted with uranyl acetateand lead citrate as previously described (King, 1999). Images werecaptured with a digital camera attached to an electron microscope (FEO10, Philips, The Netherlands). The number of regenerative clusters ofaxons at 5 weeks post-injury was determined on semithin resin section.The entire section was scored per each animal in each group. The g-ratiois the numerical ratio of unmyelinated axon diameter to myelinated axondiameter and was calculated over the entire nerve section. The frequencyof large caliber (>8 μm) myelinated fibers was calculated over theentire nerve section.

4.1.9 Statistical Analysis

Two way ANOVA with Bonferroni correction was performed to determinestatistically significant differences in the hemolytic assay (p<0.001),ELISA assay (p<0.001), SFI (p≦0.05), Footflick test (p≦0.05).

4.2 Results and Discussion

To test the effects of C activation on nerve regeneration after acutetrauma we determined the effect of C on recovery from crush injury ofthe sciatic nerve in the rat model in two complementary ways, first byexamining the effects of C6 deficiency (C6^(−/−)) and second byinhibition of C activation.

The study was set up according to a scheme that extends over a period of5 weeks. Time 0 is the time of the crush injury. Each group of animalswas treated either with placebo (PBS) or purified C6 protein or sCR1 theday before the injury (day −1) and every day thereafter until 1 weekpost-injury. Blood was collected from each animal the day before theinjury (day −1) and at days 0, 1, 2, 3, 5 and 7 post-injury to determineserum complement haemolytic activity. Functional analysis, to determinerecovery of motor function by the sciatic functional index (SFI) andrecovery of sensory function by the footflick test, was performed 1 daybefore the injury for baseline values and every week thereafter until 5weeks post-injury. Pathological analysis of the tibial nerves distalfrom the site of injury was performed at weeks 1, 3 and 5 post-injury todetermine nerve regeneration.

Both functional recovery and effects on histology were determined. Ascontrols for C6 deficiency we reconstituted C6^(−/−) rats with purifiedC6 protein (4 mg/kg/day; n=8) (C6⁺), which restored the plasma hemolyticactivity (CH₅₀) to wildtype levels (>80%; p<0.001, two way ANOVA) (Table1). Inhibition of C activation was achieved by systemic treatment withsoluble C receptor 1 (sCR1) (15 mg/kg/day; n=6), a recombinant solubleform of the human membrane C regulator CR1 which inhibits all three Cactivation pathways (Weisman et al., 1990). This treatment reducedhemolytic C activity to about 30% of the PBS vehicle-treated controls(n=6; p<0.001, two way ANOVA) over the entire course of the treatment(Table 1). We found this level of C inhibition in the plasma completelyabrogated deposition of activated C in the nerve at 3 days after injury.

Recovery of motor function was monitored every week after injury bymeasuring the sciatic functional index (SFI) calculated from the ratswalking pattern (Hare et al., 1992). At 1 week post-injury, none of theanimals used the foot of the injured leg to walk, failing to produce afootprint on the walking platform, suggesting complete loss of muscleinnervation in the leg. From week 2 post-injury and throughout the wholestudy C6^(−/−) rats (n=16) produced an SFI significantly higher thanwildtype animals (n=16; p≦0.05, two way ANOVA) (FIG. 8 a). A higher SFIresults from an increase in the print length and toe spreadingparameters and indicates re-innervation of the calf and small footmuscles, respectively. Reconstitution of the C6^(−/−) rats with purifiedC6 protein (C6+) significantly reduced the SFI (n=8; p≦0.05, two wayANOVA) to wildtype levels at week 4 and 5 post-injury. These datademonstrate that C6 deficiency in rats results in a faster recovery ofmotor function compared to wildtype. Recovery of sensory function wasassayed with the footflick test. At I week post-injury, none of theanimals retracted their paw when the footsole was stimulated by anelectric shock at 0.5 mA, suggesting complete loss of sensoryinnervation. From week 2 to week 3 post-injury, the C6^(−/−) rats showed20-50% greater recovery of sensory function compared to wildtype (n=16)and C6+ (n=8) rats (p≦0.05, two way ANOVA). The sensory function did notdiffer between groups at weeks 4 and 5 post-injury (FIG. 8 b).Similarly, animals treated with sCR1 showed a faster (10-30% increase,n=6; p<0.05, two way ANOVA) recovery of sensory function than thePBS-treated rats (n=6) between weeks 2 and 4 post-injury (FIG. 9). Thesedata indicate that both C deficiency and inhibition of C activationaccelerate and improve the return of sensory innervation to the footsoleafter sciatic crush injury.

To follow the histological regeneration of the damaged nerve, weanalyzed the tibial nerve at different time points. The regenerativeprocess is marked by the occurrence of regenerative clusters of axonswhich are sprouts of the originally injured axon. Initially, the axonsprouts reside within a single Schwann cell cytoplasm but they are laterseparated by radial sorting. Once the 1:1 relationship between Schwanncell and axon is established, the pro-myelinating SC starts to ensheaththe axon to form myelin and the basal lamina tube. At this stage, theregenerative clusters appear as groups of small caliber, thinlymyelinated axons within adjacent Schwann cells (FIG. 10, arrows). Onceone axon has reached its target, the rest of the axon sprouts areeliminated while the remaining axon increases in size. On histologicalsections at 5 weeks post-injury, untreated wildtypes and the PBSvehicle-treated controls showed regenerative clusters of small caliberthinly myelinated axons in contrast to C6^(−/−) and sCR1-treated animalswhere regenerative clusters were absent confirming a faster recoverywhen C is inhibited or absent (FIG. 10). The frequency of high caliber(>8 μm) myelinated fibers was increased in the C6^(−/−) (0.59±0.20%,n=5) and sCR1-treated (0.58±0.11%, n=6) animals compared to theuntreated wildtypes (0.05±0.02; n=5), the C6⁺ (0.06±0.01; n=3) and thePBS vehicle-treated (none; n=6) controls at 5 weeks post-injury whereasno difference in the frequency of low (<4 μm) and intermediate (4-8 μm)caliber myelinated fibers was found. The myelin thickness was notaltered between groups of animals (g-ratio of 0.69±0.01, n=5, wildtype;0.65±0.02, n=5, C6^(−/−); 0.65±0.01, n=3, C6⁺; 0.70±0.01, n=6,sCR1-treated; 0.66±0.003, n=6, PBS vehicle-treated) (data not shown).

Taken together, these data show that axonal regeneration and functionalrecovery after peripheral nerve injury are enhanced in the absence of C6or when C activation is inhibited by sCR1. Thus the ability to form MACis a negative determinant of nerve recovery.

Functional recovery after axonal crush injury requires axons to re-enterthe Schwann cell tubes injured at the crush site. Once in the distalstump, the axons need to re-navigate the paths followed before injuryand generate specific synapse with exactly the same muscle fiber theyhad previously innervated. In this task they are guided by attractiveand repulsive molecular cues (Tessier-Lavigne and Goodman, 1996; Yu andBargmann, 2001) but recent evidence showed that physical factors alsoplay a key role (Nguyen et al., 2002). Thus, maintenance of intactendoneurial tubes could be of high importance for the regenerating adultperipheral nerve.

Blockade of C activation, and particularly MAC formation, reduces tissuedamage during nerve degeneration, appears to rescue the architecturenecessary for the guidance of the axon and resulting in more efficientregeneration and recovery of function. Functional improvement in theabsence of increased myelin sheath thickness can be explained by theincrease in the number of large caliber fibers.

A wealth of evidence over the last decade points to a possiblebeneficial role of macrophages during recovery (Kiefer et al., 2001).Later after injury, macrophages secrete anti-inflammatory cytokineswhich are involved in resolving the inflammatory process. Once theinflammation terminates, macrophages contribute to Schwann cellproliferation and survival, remyelination and recovery through thesecretion of growth and differentiation factors. We have shown that,early after injury, C inhibition markedly reduces infiltration ofendoneurial macrophages (5-fold increase, compared to 25-fold in theabsence of C inhibition) (Ramaglia et al., 2007). We postulated thatthis is due to the proliferation of the resident macrophage populationwhile little contribution comes from the haematogenous macrophages. Itis possible that we separated the detrimental effect of thehaematogenous macrophages from the beneficial effect which can beexerted by the endoneurial population.

Our findings open the door to a novel therapeutic approach in whichblockade of the C cascade, or selective inhibition of MAC, promotesregeneration after traumatic injury and in peripheral neuropathies andneurodegenerative diseases where C-dependent nerve damage has beenreported.

TABLE 1 Plasma haemolytic activity (% CH50). Day 0 Day −1 (crush) Day 1Day 2 Day 3 Day 4 Day 5 Day 7 Wildtype 91.6 ± 1.0 91.7 ± 1.1  81.2 ±1.7  89.5 ± 1.5  86.1 ± 1.4  n.d. 82.1 ± 1.7  90.8 ± 4.1  PBS-treated C6deficient 14.0 ± 0.1 n.d. n.d. 12.8 ± 0.2  n.d. 13.5 ± 0.3  n.d. 15.7 ±0.4  PBS-treated C6 deficient 14.0 ± 0.1 n.d. n.d. 78.5 ± 0.9* n.d. 76.9± 0.8* n.d. 78.5 ± 1.6* C6-treated (C6⁺) Wildtype 87.4 ± 0.6 36.8 ± 1.1*27.2 ± 0.9* 27.2 ± 3.6* 27.9 ± 0.3* n.d. 29.6 ± 1.7* 33.4 ± 29*sCR1-treated C dependent hemolysis in serum from wildtype PBS-treated,C6^(−/−) PBS-treated and C6^(−/−) reconstituted with purified human C6(C6⁺) rats and sCR1-treated rats. Treatment started 1 day (day −1)before the injury (day 0) and it was repeated every day until 1 week.Plasma was collected at days −1, 2, 4, and 7 immediately before thetreatment. Values are means ± S.D. of six to eight animals per group pertime point. Statistical significance (*) refers to p ≦ 0.001 determinedby a two way ANOVA test with Bonferroni correction. n.d., notdetermined.

Example 5 5.1 Materials and Methods 5.1.1 Animals

This study was approved by the Academic Medical Center Animal EthicsCommittee and complies with the guidelines for the care of experimentalanimals.

Male 12 weeks old PVG/c were obtained from Harlan (UK). The animalsweighed between 200 g and 250 g and were allowed acclimatization for atleast two weeks before the beginning of the study. Animals were kept inthe same animal facility during the entire course of the experiment andmonitored for microbiological status according to the FELASArecommendations. Animals were housed in pairs in plastic cages. Theywere given rat chow and water ad libitum and kept at a room temperatureof 20° C. on a 12 hours:12 hours light:dark cycle.

5.1.2 Administration of sCR1 or Cetor for Inhibition Studies

Recombinant soluble complement receptor I (sCR1) was obtained aspreviously described (Piddlesden et al., 1994). Complement C1 inhibitor(Cetor) was kindly provided by Sanquin (Amsterdam, The Netherlands).sCR1 was administered i.p. in twelve (12) rats at a dose of 15mg/kg/day. Cetor was administered i.v. in six (6) rats at a dose of 50U/rat/day. Twelve (12) rats were treated with equal volumes of vehicle(PBS) alone. The treatment was given one day before the crush injury(day −1) and every 24 hours (day 0, 1, 2) until the nerves were removedat 3 days post-injury. Ten (10) rats were treated either with sCR1 (6)or with PBS (4) up to 6 days post-injury (day −1, 0, 1, 2, 3, 4, 5, 6)and the nerves were removed 1 day after the end of the treatment (day7).

5.1.3 Hemolytic Assay and ELISA

Blood samples from PBS- and sCR1-treated rats were collected from thetail vein one day before the crush injury (day −1) and every followingday (day 0, 1, 2) until the animals were sacrificed at 3 days after theinjury. In the group treated up to 6 days, additional blood samples werecollected at day 3, 5 and 7 after injury. All samples were collectedimmediately before each injection of treatment. Plasma was separated andstored at −80° C. until used to monitor sCR1 inhibitory activity viastandard complement hemolytic assay (Morgan, 2000).

Plasma levels of sCR1 were measured using ELISA assay as previouslydescribed (Mulligan et al., 1992) using serial dilutions assayed induplicates.

5.1.4 Nerve Crush Injury and Tissue Processing

All the surgical procedures were performed aseptically under deepisoflurane anesthesia (2.5% Vol isoflurane, 1 L/min O₂ and 1 L/min N₂O).The left thigh was shaved and the sciatic nerve was exposed via anincision in the upper thigh. The nerve was crushed for three 10 speriods at the level of the sciatic notch using smooth, curved forceps(No. 7). The crush site was marked by a suture which did not constrictthe nerve. On the right side, a sham surgery was performed which exposedthe sciatic nerve but did not disturb it. A suture was also placed. Themuscle and the skin were closed with stitches. Following the crush, therats were allowed to recover for 3 days (PBS-treated n=8; sCR1-treatedn=6; Cetor-treated n=6) and 7 days (PBS-treated n=4; sCR1-treated n=6).

All the animals were intracardially perfused with 4% paraformaldehyde inpiparazine-N—N′-bis(2-ethane sulphonic acid) (PIPES) buffer (pH 7.6).Left and right sciatic nerves were removed from each animal and onesegment of 5 mm length was collected distally from the crush site. Eachsegment was conventionally processed into paraffin wax forimmunohistochemistry.

5.1.5 Immunohistochemistry

Paraffin wax sections were stained using a three-step immunoperoxidasemethod. All the incubations were performed at room temperature (RT).Following deparaffination and rehydration, endogenous peroxidaseactivity was blocked with 1% H₂O₂ in methanol for 20 min. In all cases,microwave antigen retrieval was used (800 W for 3 min followed by 10 minat 440 W in 10 mM Tris/1 mM EDTA pH 6.5). To block the non-specificbinding sites, slides were incubated in 10% normal goat serum (NGS) inTris buffered saline (TBS) for 20 min. Following incubation in theappropriate primary antibody diluted in 1% BSA (see Table 2) for 90 min,sections were incubated for 30 min in biotinylated goat anti-rabbit orgoat anti-mouse IgG from DakoCytomation (Glostrup, DK) diluted 1:200 in1% BSA and 30 min in horseradish peroxidase labeled polystreptavidin(ABC-complex, DAKO). To visualize peroxidase activity, the slides wereincubated in 0.05% 3-amino-9-ethylcarbazole in acetate buffer (pH 5) for5 min followed by a 30 sec counterstaining with hematoxylin and mountedin gelatin. Sections immunostained with secondary conjugate alone wereincluded with every experiment as negative controls while sections ofrat spinal cord and lymph nodes served as positive controls.

Images were captured with a digital camera (Olympus, DP12, TheNetherlands) attached to a light microscope (Olympus, BX41, TheNetherlands).

TABLE 2 Antibodies, source and dilutions for immunohistochemistry. Dilu-Antibodies Source tions Monoclonal mouse anti-human Stemberger 1:1000Phosphorilated neurofilament (Lutherville, UK) (SMI31 clone) Polyclonalrabbit anti-human MBP DakoCytomation 1:100 (Glostrup, DK) Monoclonalmouse anti-rat CD68 Serotec (Oxford, UK) 1:100 (ED1 clone) Polyclonalrabbit anti-rat C9 B. P. Morgan 1:300 Polyclonal rabbit anti-human C3cDakoCytomation 1:750 (Glostrup, DK) Polyclonal rabbit anti-human C4cDakoCytomation 1:100 (Glostrup, DK)

5.1.6 Quantitative Analysis of Immunohistochemistry

All analyses were performed with the Image Pro Plus version 5.02 (MediaCybernatics, The Netherlands). CD68 (ED1 clone)-immunoreactive cellswere scored positive when the CD68 positive signal was associated withnuclei. Thirty non-consecutive sections of sciatic nerve per rat werescored. An average of 3 non-overlapping fields of view including >90% ofthe entire nerve area was taken for each section. Quantification of theMAC and MBP immunostaining was performed at 40× magnification on twonon-overlapping fields per section examined. Ten sections per rat werescored. The surface area stained is expressed as percentage of totalarea examined.

5.1.7 Protein Extraction and Western Blot Analysis

Frozen sciatic nerves from 2 untreated rats sacrificed at 2 daysfollowing the crush injury were homogenized using a pestle and mortar inliquid nitrogen in 20 mmol l⁻¹ Tris (pH 7.4), 5 mmol l⁻¹1,4-dithio-DL-threitol (DTT) and 0.4% SDS and 6% glycerol. Thehomogenates were centrifuged at 10,000×g, at 2° C. for 10 min. Thesupernatant fraction was collected and used for protein analysis.Protein concentrations were determined with a DC protein assay kit(Bio-Rad Laboratories, USA), using bovine serum albumin (BSA) as astandard.

Protein extracts (20 μg/sample) were boiled for 5 min, separated by 10%SDS-PAGE and transferred to nitrocellulose membrane overnight at 4° C.Prior to blotting, the nitrocellulose membranes were stained withPonseau red for 30 sec to verify protein load. The membranes werepre-incubated in 50 mmol l⁻¹ TrisHCl containing 0.5% Tween20 (TBST) and5% non-fat dried milk for 1 hour at RT. Blots were incubated for 2 hoursin the polyclonal goat anti-factor Bb (fBb) (Quidel, San Diego, Calif.)diluted in TBST containing 5% non-fat dried milk. Following washing inTBST, the membranes were incubated for 1 hour in polyclonal rabbitanti-goat horseradish peroxidase-conjugated secondary antibody diluted1:2000 in TBST containing 5% non-fat dried milk. Membranes were washedin TBST for 30 min and immunoreactive bands were detected using enhancedchemiluminescence (ECL, Amersham, Piscataway, N.J., USA). Quantificationof the immunoreactive bands was performed using Advanced Image DataAnalyzer software v. 3.4 (Raytest, Germany).

5.1.7 Statistical Analysis

Two-way ANOVA with Bonferroni correction was performed to determinestatistically significant differences (p≦0.001). Statistical analysis ofthe immunoblotting quantification was determined by unpaired t-test(p≦0.05).

5.2 Results

5.2.1 sCR1 Blocks Complement Activation after Acute Nerve Trauma

To determine the effects of inhibition of all complement activationpathways on Wallerian degeneration (WD), we treated animals with sCR1.Treatment was started 1 day prior to a crush injury of the sciaticnerve. We measured plasma sCR1 levels and CH50 after daily i.p.injections of either sCR1 at a dose of 15 mg/kg/day or equal volume ofvehicle. sCR1 levels increased after the first day of injection andhemolytic complement activity was reduced to about 30% of controls (FIG.11).

The sCR1 treated animals showed inhibition of complement activation inthe crushed nerve (FIG. 12). The sCR1-treated nerves showed virtually noMAC deposits (0.8±0.9%) whereas MAC immunoreactivity covered 31.4±7.8%of the total area examined in the nerves of the PBS-treated rats. MACimmunoreactivity was undetectable in the uninjured control nerves.Deposition of C4c and C3c was also prevented in the sCR1-treated nerveswhereas high amount of immunoreactivity was detected in the PBS-treatednerves (not shown). These results demonstrate that sCR1 is an effectiveinhibitor of complement activation after acute nerve trauma.

5.2.2 sCR1 Protects Nerves from Axon Loss at 3 Days Post-Injury

To determine the effects of sCR1-mediated complement inhibition on WDmorphological changes of axons and myelin at 3 days post-injury wereanalyzed.

Neurofilament (SMI31) staining showed that the sciatic nerve ofPBS-treated rats had empty and enlarged axonal spaces, delimited by athin immunoreactive axolemma, and sparse axonal debris within the nervewhich are signs of axonal swelling and degradation (data not shown). Incontrast, the sCR1-treated rats still showed the typical punctuatedappearance of axons, similarly to the uninjured control nerve,demonstrating rescued axonal breakdown at 3 days after injury. Myelin(MBP) immunostaining revealed signs of myelin breakdown in nerves ofPBS-treated rats at 3 days following the injury whereas the nerves ofsCR1-treated rats showed the typical annulated myelin staining similarto uninjured control nerves, demonstrating rescued myelin degradation atthis time point after injury (data not shown). These observationsdemonstrate that sCR1 protects nerves from axonal degradation and myelinat 3 days post-injury.

Analysis of sciatic nerves of both PBS- and sCR1-treated rats at 7 dayspost-injury shows axonal and myelin breakdown in both groups of animals,demonstrating that WD was delayed but not prevented in sCR1-treatednerves following the crush injury (data not shown).

Quantification of the MBP staining showed significant lowerimmunoreactivity in the crushed nerves compared to the uninjured nerves(21.7±3.5%). The amount of MBP immunoreactive debris differed betweennerves of PBS- and sCR1-treated rats. The PBS-treated nerves showedsignificantly less percentage of MBP immunoreactive area (2.1±1.3%)compared to the sCR1-treated nerves (7.6±1.0%). This demonstrates thatclearance of myelin debris is delayed in the sCR1-treated nerves.

5.2.3 sCR1 Prevents Macrophage Accumulation and Activation at 3 DaysPost-Injury

We monitored accumulation and morphological changes of macrophagesbecause complement activation mediates macrophages recruitment andactivation. We used the CD68 antibody (ED1 clone), a lysosomal marker,as marker for their metabolic state. A few CD68 immunoreactive cellswere found in the control uninjured nerve (5.3±1.7 cells/mm²). Thenumber increased to 261.2±10.7 cells/mm² in the nerves of thePBS-treated rats at 3 days post-injury while the nerves from thesCR1-treated rats showed a milder increase (63.1±4.7 cells/mm²) (FIG.13).

The nerves of the PBS-treated rats showed large and asymmetrical CD68immunoreactive cells (average size 103.6±71.8 μm²) at 3 dayspost-injury, while small and round cells (average size 22.8±14.1 μm²)were detected in the nerves of the sCR1-treated rats, a size and shapesimilar to that seen in the uninjured control nerves (average size18.8±6.6 μm²) (data not shown).

Determination of the CD68 immunoreactive cell size distribution wasperformed on 11 cells in the uninjured nerves, 778 cells in thePBS-treated nerves and 294 cells in the sCR1-treated nerves. Cell sizedistribution showed high variability in the PBS-treated nerves with celldimension ranging from 20 to more than 400 μm² with a large populationof cells of about 60 μm². In contrast, the sCR1-treated nerves showedcell dimension ranging from 0 to 40 μm², similar to the size of cellsfound in the uninjured control nerves (FIG. 14). The colocalization ofMBP and CD68 shows macrophages engulfing myelin in the PBS-treatednerves while small resting macrophages are visible between themorphologically intact myelin sheaths of the uninjured and sCR1-treatednerves (data not shown). These results show that macrophages areactivated in the PBS-treated nerves but not in the sCR1-treated ones.

5.2.4 Activation of the Alternative Pathway after Acute Nerve Trauma

We have found that the classical pathway of the complement system isactivated after acute nerve trauma. To determine whether the alternativepathway is also triggered by a crush injury of the sciatic nerve, wemeasured the expression level of Bb, the 60 kD protein fragment whichresults from the cleavage of factor B. Low levels of Bb immunoreactivitywere detected in protein extracts of uninjured rat nerves, whereas anear two fold increase (1.8±0.2) was seen at 2 days following the crushinjury (FIG. 15 A, B). These results indicate that the alternativepathway loop is triggered after acute nerve trauma, generating morecleaved fB.

5.2.5 Effects of C1 Inhibitor on WD

To determine whether the alternative pathway is sufficient to causepathology we treated rats with C1 inhibitor (Cetor). Inhibiting theclassical and lectin pathways with the complement C1 inhibitor, Cetor,would allow us to determine the contribution of the different complementpathways. Cetor dosage was extrapolated from the work of de Smet et al.Immunostaining of the Cetor treated crushed nerves for activationproducts of the classical pathway (C4c) was negative and thus suggestedinhibition of the classical pathway.

Low amounts of MAC immunoreactivity (7.3±2.7% of total area examined)were visible in the nerves of Cetor-treated animals at 3 dayspost-injury and the staining was mainly localized in the axonalcompartment of some fibers (data not shown). The neurofilament (SMI31clone) staining showed fibers with normal punctuated axonalimmunoreactivity and fibers with atypical annulated immunoreactivityoutlining enlarged axonal spaces. This demonstrates abnormaldistribution of the phosphorylated neurofilament epitope, compatiblewith neurofilament breakdown (data not shown). These observationssuggest an intimate link between MAC deposition and axon loss. Themyelin (MBP) staining showed normal annulated myelin morphology (datanot shown) and the CD68 staining revealed a number of cells (59.8±28.3cells/mm²) similar to that observed in the sCR1-treated nerves. Inaddition the average CD68 immunoreactive cell size (19.1±10.5 μm²) andsize distribution determined on 218 cells did not differ fromsCR1-treated nerves or uninjured controls (data not shown). Thecolocalization of MBP and CD68 shows small resting macrophages betweenthe morphologically intact myelin sheaths (data not shown). Theseresults suggest a link between lack of macrophage activation andpreserved myelin morphology at 3 days post-injury.

5.3 Discussion

The present invention demonstrates that systemic treatment with sCR1, aninhibitor of classical, lectin and alternative pathways of complementactivation, protects from early axon loss and myelin breakdown afterperipheral nerve injury.

Daily administration of sCR1 to injured rats prevented both systemic andlocal complement activation, resulting in blockade of MAC deposition inthe nerve. In untreated animals, crush injury leads to a rapid increaseof CD68 positive cells which enlarge and phagocytose myelin. In theinhibitor-treated nerves only a slight increase of CD68 positive cellswas detectable but they failed to enlarge. This appears to be due to theproliferation and differentiation of the endoneurial macrophagepopulation which occurs already at 2 days after injury (Mueller et al.,2001). Both long-term and short-term resident macrophages newly expressthe lysosomal ED1 antigen and have the potential to phagocytose myelin(Leonhard et al., 2002). However, this is a complement-mediated event(Bruck W and Friede, 1991). Since complement activation is inhibited inthe sCR1-treated nerves, complement opsonins are not deposited on thenervous tissue hampering target recognition and preventing myelinphagocytosis. In addition, complement inhibition also results ininefficient chemotaxis, preventing the recruitment of blood-derivedmacrophages which probably accounts for the additional 4 fold increaseobserved in the PBS-treated nerve.

Despite the diminished recruitment and activation of macrophages, sCR1cannot protect the nerve from axonal degradation and myelin breakdown at7 days post-injury even when hemolytic complement activity is maintainedlow. Therefore we conclude that inhibition of complement activation onlyaffects the early events of WD. Lack of C4c deposition in thesCR1-treated nerves is a noteworthy finding because sCR1 inhibits the C3convertase which is downstream of C4 cleavage, thus little effect on C4cdeposition would be expected. However, as also noted in previous studies(Piddlesden et al., 1994), blockade of C-mediated damage by sCR1 willalso inhibit overall C deposition on damaged tissue, also resulting inundetectable C4c levels.

We demonstrated that, beside the classical pathway, also the alternativepathway is activated following a crush injury of the peripheral nerve.Blockade of the classical (and lectin) pathway of complement with C1inhibitor (Cetor), a serine protease inhibitor which blocks activationof the C1q-C1r-C1s (and MBL-MASP) complex,^(6,10) diminished but did notablate MAC deposition in the nerve. Since low rate activation of thealternative pathway occurs under physiological conditions and isnegatively regulated by complement inhibitors, disruption of membranebound complement regulatory components at the site of injury could setthe alternative pathway out of control, generating more C3 convertaseand leading to MAC deposition. In addition, we cannot rule out that lowlevels of C3b, which would accumulate during activation of the classicalpathway, could escape inhibition by Cetor forming low levels of C5convertase and acting as substrate for the alternative pathway tofurther amplify activation. Partial blockade of complement activationresults in reduced C3 deposition which reduces macrophage accumulationand prevents their activation while low amounts of MAC are stilldeposited in the nerve. Interestingly, this is sufficient to causemarked axonal injury (but not much myelin degradation), emphasizing thesensitivity of the axons to MAC-induced damage. This also suggests thatmyelin loss is an indirect effect of axon loss and it requiresmacrophages to target the opsonised surface, become activated, strip anddegrade the myelin.

Our data show that even low levels of MAC deposition, occurring with C1inhibitor treatment, are sufficient to cause marked axonal damage.

This invention demonstrates that C-inhibitors protect the peripheralnerve from early axonal degradation and myelin breakdown due to amechanical injury. Previous studies on demyelinating diseases of thePNS, such as Guillan Barré Syndrome, have been performed on animalmodels immunized with peripheral nerve myelin to induce the diseasephenotype, making their findings directly applicable to diseases wherean antigen-antibody complex is likely to mediate complement activation.In WD of the peripheral nerve after a crush injury, complementactivation occurs in an antibody-independent manner, directly targetingepitopes on damaged axons and myelin. Thus, the data show thatC-inhibitors are also promising tools in the treatment of non-autoimmunediseases, such as inherited peripheral neuropathies, where a secondaryrole of the immune system superimposed to the primary genetic defect hasrecently emerged (reviewed in Martini R and Toyka, 2004).

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1-14. (canceled)
 15. A method of treating a subject having a conditionrequiring axonal regeneration comprising administering to the subject atherapeutically effective amount of an inhibitor of a mammaliancomplement system.
 16. The method according to claim 15, wherein thecondition requiring axonal regeneration is a physical injury of aperipheral nerve.
 17. The method according to claim 15, wherein thecondition requiring axonal regeneration is a neurodegenerative disorderof the peripheral or central nervous system.
 18. The method according toclaim 17, wherein the condition is Amyotrophic Lateral Sclerosis (ALS).19. The method according to claim 17, wherein the condition isHereditary Motor and Sensory Neuropathy (HMSN).
 20. The method accordingto claim 16, wherein the condition is Huntington's Disease (HD).
 21. Themethod according to claim 16, wherein the condition is injury of theCNS.
 22. The method according to claim 16, wherein the condition isinjury of the PNS.
 23. The method according to claim 15, wherein theinhibitor inhibits formation of a membrane attack complex.
 24. Themethod according to claim 15, wherein the inhibitor is an antibody thatblocks one or more of C3 convertase, C5, C6, C7, C8, C9, or MACassembly.
 25. The method according to claim 24, wherein the antibody isa human or humanized antibody.
 26. The method according to claim 15,wherein the inhibitor is selected from the group consisting of anantisense oligonucleotide, a complement regulator, a complementreceptor, an antibody or antibody-fragment against a complementcomponent, cobra venom factor, a poly-anionic inhibitor of complement,K-76COOH, rosmaric acid, nafamastat mesilate, C1s-INH-248, compstatin,PMX53, and PMX205.
 27. The method according to claim 17, wherein theinhibitor inhibits C6.
 28. The method according to claim 17, wherein theinhibitor is an antibody or an antisense oligonucleotide.
 29. The methodaccording to claim 15, wherein the inhibitor is administered at or neara site of injury.
 30. The method of claim 15, wherein the inhibitor isadministered by infusion, bolus injection, or parenterally.
 31. Themethod of claim 15, wherein the inhibitor is administered by anintradermal, intramuscular, intraperitoneal, intravenous, orsubcutaneous route.